Molecular mechanisms coordinating functional and morphological plasticity at the synapse: Role of GluA2/N-cadherin interaction-mediated actin signaling in mGluR-dependent LTD

Long-lasting synaptic plasticity involves changes in both synaptic morphology and electrical signaling (here referred to as structural and functional plasticity). Recent studies have revealed a myriad of molecules and signaling processes that are critical for each of these two forms of plasticity, but whether and how they are mechanistically linked to achieve coordinated changes remain controversial.It is well accepted that functional plasticity at the excitatory synapse is dependent upon the activities of glutamate receptors. While the activation of NMDARs (N-methyl-D-aspartic acid receptors) and/or mGluRs (metabotropic glutamate receptors) is required for the induction of many forms of plasticity, AMPARs (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors), the principal mediators of fast excitatory synaptic transmission, are the ultimate targets of modifications that express functional plasticity. Investigations exploring structural plasticity have been mainly focused on the small membranous protrusions on the dendrites called spines. The morphological regulation of these spines is mediated by the reorganization of the actin cytoskeleton, the predominant structural component of the synapse. In this regard, the Rho family of GTPases, particularly Rac1, RhoA and Cdc42, is found to be the central regulator of spine actin and structural plasticity of the synapse.It is thought that the collaborative interaction between functional and structural factors underlies the sustained or permanent nature of long-lasting synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD), the most extensively studied forms of synaptic plasticity widely regarded as cellular mechanisms for learning and memory. However, data specifically pertaining to whether and how these two distinct components are linked at the molecular level remain sparse. In this regard, we have identified a number of synaptic proteins that are involved in both structural and functional changes during mGluR-dependent LTD (mGluR-LTD). Among these are the GluA2 (formerly called GluR2) subunit of AMPARs, Rac1 and Rac1-activated kinases. We have discovered that these proteins interact and reciprocally regulate each other, which led us to hypothesize that the GluA2–Rac1 interaction may serve as a coordinator between functional and morphological plasticity. In this review, we will briefly discuss the available evidence to support such a hypothesis.     

Comparative expression analysis of senescence gene CsNAP and B-class floral development gene CsAP3 during different stages of flower development in Saffron (Crocus sativus L.)

Physiology and Molecular Biology of Plants - Crocus sativus, a monocot triploid species belonging to the Iridaceae family, is cultivated for its red stigmatic lobes of the carpel that constitute...     

Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression

We studied dynamics of cell surface expression ofproteolytically activated thrombin receptor (PAR-1) in human pulmonaryartery endothelial cells (HPAEC). PAR-1 activation was measured bychanges in cytosolic calcium concentration([Ca²⁺]_i)and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance).[Ca²⁺]_iincrease in response to thrombin was abolished by preexposure to 25 nMthrombin for >60 min, indicating PAR-1 desensitization, butpreexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin forup to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombindecreased monolayer electrical resistance 40-60%. Cellspreexposed to 10 nM thrombin, but not those preexposed to 25 nMthrombin, remained responsive to thrombin 3 h later. Loss of cellretractility was coupled to decreased cell surface PAR-1 expression asdetermined by immunofluorescence. Cell surface PAR-1 disappeared uponshort-term (30 min) thrombin exposure but reappeared within 90 minafter incubation in thrombin-free medium. Exposure to 25 nM thrombinfor >60 min prevented rapid cycloheximide-insensitive PAR-1reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1expression required 18 h. Therefore, both duration and concentration ofthrombin exposure regulate the time course of recovery of HPAEC surfacePAR-1 expression. The results support the hypothesis that initialrecovery of PAR-1 surface expression in endothelial cells results froma rapidly mobilizable PAR-1 pool, whereas delayed recovery results fromde novo PAR-1 synthesis. We conclude that thrombin itself regulatesendothelial cell surface PAR-1 expression and that decreased surfaceexpression interferes with thrombin-induced endothelial cell activation responses.

     

Chain termination in polyhydroxyalkanoate synthesis: involvement of exogenous hydroxy-compounds as chain transfer agents.

We have identified a range of compounds which, when present during poly(3-hydroxybutyrate) [P(3HB)] accumulation by Ralstonia eutropha (reclassified from Alcaligenes eutrophus), can act as chain transfer agents in the chain termination step of polymerization. End-group analysis by 31P NMR of polymer derivatized with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane revealed that all these compounds were covalently linked to P(3HB) at the carboxyl terminus. All chain transfer agents possessed one or more hydroxyl groups, and glycerol was selected for further investigation. The number-average molecular mass (Mn) of P(3HB) produced by R. eutropha from glycerol was substantially lower than for polymer produced from glucose, and we identified two new end-group structures. These were attributed to a glycerol molecule bound to the P(3HB) chain via the primary or secondary hydroxyl groups. When a primary hydroxyl group of glycerol is involved in chain transfer, the end-group structure is in both [R] and [S] configurations, implying that chain transfer to glycerol is a random transesterification and that PHA synthase does not catalyse chain transfer. 3-Hydroxybutyric acid is the most probable chain transfer agent in vivo, with propagation and termination reactions involving transfer of the P(3HB) chain to enzyme-bound and free 3-hydroxybutyrate, respectively. Only carboxyl end-groups were detected in P(3HB) extracted from exponentially growing bacteria. It is proposed that a compound other than 3-hydroxybutyryl-CoA acts as a primer in the initiation of polymer synthesis.     

Role of NF-kappaB-dependent caveolin-1 expression in the mechanism of increased endothelial permeability induced by lipopolysaccharide

We investigated the role of NF-kappaB activation by the bacterial product lipopolysaccharide (LPS) in inducing caveolin-1 (Cav-1) expression and its consequence in contributing to the leakiness of the endothelial barrier. We observed that LPS challenge of human lung microvascular endothelial cells induced concentration- and time-dependent increases in expression of Cav-1 mRNA and protein. The NEMO (NF-kappaB essential modifier binding domain)-binding domain peptide (IkB kinase (IKK)-NEMO-binding domain (NBD) peptide), which prevents NF-kappaB activation by inhibiting the interaction of IKKgamma with the IKK complex, blocked LPS-induced Cav-1 mRNA and protein expression. Knockdown of NF-kappaB subunit p65/RelA expression with small interfering RNA also prevented LPS-induced Cav-1 expression. Caveolae open to the apical and basal plasmalemma of endothelial cells increased 2-4-fold within 4 h of LPS exposure. IKK-NBD peptide markedly reduced the LPS-induced increase in the number of caveolae as well as transendothelial albumin permeability. These observations were recapitulated in mouse studies in which IKK-NBD peptide prevented Cav-1 expression and interfered with the increase in lung microvessel permeability induced by LPS. Thus, LPS mediates NF-kappaB-dependent Cav-1 expression that results in increased caveolae number and thereby contributes to the mechanism of increased transendothelial albumin permeability.     

Critical role of Cdc42 in mediating endothelial barrier protection in vivo

Activation of the Rho GTPase Cdc42 has been shown in endothelial cell monolayers to prevent disassembly of interendothelial junctions and the increase in endothelial permeability. Here, we addressed the in vivo role of Cdc42 activity in mediating endothelial barrier protection in lungs by generating mice expressing the dominant active mutant V12Cdc42 protein in vascular endothelial cells targeted via the VE-cadherin promoter. These mice developed normally and exhibited constitutively active GTP-bound Cdc42. The increase in lung vascular permeability and gain in tissue water content in response to intraperitoneal lipopolysaccharide challenge (7 mg/kg) were markedly attenuated in the transgenic mice. To address the basis of the protective effect, we observed that expression of V12Cdc42 mutant in endothelial monolayers reduced the decrease in transendothelial electrical resistance, a measure of opening of interendothelial junctions, thus indicating that Cdc42 activity preserved junctional integrity. RhoA activity in V12Cdc42-expressing endothelial monolayers was reduced compared with untransfected cells, suggesting that activated Cdc42 functions by counteracting the canonical RhoA-mediated mechanism of endothelial hyperpermeability. Therefore, Cdc42 activity of microvessel endothelial cells is a critical determinant of junctional barrier restrictiveness and may represent a means of therapeutically modulating increased lung vascular permeability and edema formation.     

Thrombin receptors activate G(o) proteins in endothelial cells to regulate intracellular calcium and cell shape changes

Thrombin receptors couple to G(i/o), G(q), and G(12/13) proteins to regulate a variety of signal transduction pathways that underlie the physiological role of endothelial cells in wound healing or inflammation. Whereas the involvement of G(i), G(q), G(12), or G(13) proteins in thrombin signaling has been investigated extensively, the role of G(o) proteins has largely been ignored. To determine whether G(o) proteins could contribute to thrombin-mediated signaling in endothelial cells, we have developed minigenes that encode an 11-amino acid C-terminal peptide of G(o1) proteins. Previously, we have shown that use of the C-terminal minigenes can specifically block receptor activation of G protein families (). In this study, we demonstrate that G(o) proteins are present in human microvascular endothelial cells (HMECs). Moreover, we show that thrombin receptors can stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to G(o) proteins when co-expressed in Sf9 membranes. The potential coupling of thrombin receptors to G(o) proteins was substantiated by transfection of the G(o1) minigene into HMECs, which led to a blockade of thrombin-stimulated release of [Ca(2+)](i) from intracellular stores. Transfection of the beta-adrenergic kinase C terminus blocked the [Ca(2+)](i) response to the same extent as with G(o1) minigene peptide, suggesting that this G(o)-mediated [Ca(2+)](i) transient was caused by Gbetagamma stimulation of PLCbeta. Transfection of a G(i1/2) minigene had no effect on thrombin-stimulated [Ca(2+)](i) signaling in HMEC, suggesting that Gbetagamma derived from G(o) but not G(i) could activate PLCbeta. The involvement of G(o) proteins on events downstream from calcium signaling was further evidenced by investigating the effect of G(o1) minigenes on thrombin-stimulated stress fiber formation and endothelial barrier permeability. Both of these effects were sensitive to pertussis toxin treatment and could be blocked by transfection of G(o1) minigenes but not G(i1/2) minigenes. We conclude that the G(o) proteins play a role in thrombin signaling distinct from G(i1/2) proteins, which are mediated through their Gbetagamma subunits and involve coupling to calcium signaling and cytoskeletal rearrangements.